It is widely accepted that regulatory T cells (Tregs) are critical for the prevention of autoimmunity and maintenance of self-tolerance throughout the lifespan of a human being. A diminished frequency or dysfunction of Tregs has been reported in many human diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis, type 1 diabetes, multiple sclerosis (MS), aplastic anemia, idiopathic thrombocytopenic purpura, and graft-versus-host disease (GVHD), as well as transplant rejection. Due to questions surrounding the purity of the Tregs used in these studies, however, the role of Tregs in the pathogenesis of these human diseases remains unclear.
In contrast, the evidence is strong regarding the utility of Tregs for the treatment of autoimmunity in murine models. In murine studies, adoptive immunotherapy with Tregs has been shown to be effective in the prevention of experimental autoimmune encephalomyelitis (EAE), type 1 diabetes, SLE, autoimmune gastritis, inflammatory bowel disease (IBD), asthma, aplastic anemia, graft rejection, and GVHD.
Currently, enhancing graft acceptance and preventing GVHD are probably the most fruitful areas for the application of Treg immunotherapy in humans and a related condition that might greatly benefit from Treg immunotherapy is chronic GVHD (cGVHD). Approximately 30-50% of patients receiving allogeneic HSCT will develop cGVHD, which can be life threatening or can severely impair the patient's quality of life. With improvement in postgrafting immunosuppressive regimens, the number of individuals at risk for cGVHD is increasing. Treatment remains unsatisfactory and corticosteroids are still the mainstay of therapy. The pathophysiology and molecular mechanisms of this disease are still unclear. However, the loss of self-tolerance resulting in autoimmune manifestations is a major component.
Thus, a source of regulatory T cells for the treatment of autoimmunity and transplant rejection via adoptive immunotherapy in humans is urgently needed to advance the treatment of these prevalent but poorly treated diseases.
One aspect of Treg physiology that has not been considered is whether Tregs develop a normal T-cell receptor (TCR) repertoire after HSCT. Regulatory T cells develop in the thymus and their T-cell receptor (TCR) repertoire is shaped by interaction with thymic epithelial or dendritic cells. Tregs have been shown to express a diversified TCR repertoire similar to, but also distinct from, that of CD4+FOXP3− conventional T cells. As the thymus microenvironment in patients who have received myeloablative treatment may be compromised, a factor contributing to the development of cGVHD is the development and selection of an abnormal or skewed TCR repertoire in Tregs, compared with conventional T cells. A loss of diversity of TCR expression in the donor's Treg population results in an inability of the Tregs to inhibit host-reactive effector cells, leading to the development of cGVHD.
The study of human Tregs and their use in therapy has been hampered by the inability to obtain sufficient numbers of these cells for cellular immunotherapy. There is a great need to overcome the inability to consistently achieve a highly purified FOXP3+ Treg product after ex vivo expansion, secondary to the outgrowth of contaminating non-Tregs.
While a CD4+FOXP3− population of more than 90% purity can be isolated by fluorescence-activated cell sorting (FACS) of the top 2-4% of CD4+ T cells with low levels of CD127 and high levels of CD25 expression (CD127lowCD25hi) from peripheral blood, the percentage of FOXP3+ T cells decreases to 75% after 1 week and to 50% after 2 weeks of in vitro expansion.
Different strategies have been developed to optimize the purity of Tregs. One method is the addition of rapamycin to the expansion cultures. This is based on the evidence that FOXP3− T cells are more susceptible to rapamycin-induced apoptosis than FOXP3+ T cells. But it remains difficult to obtain populations of expanded FOXP3+ with purity greater than 90% after 14 days of expansion using this technique and thus, the large variations in purity remain an issue.
The use of CD4+CD45RA+CD25hi Tregs as the starting population has also been advocated to improve the purity of Tregs during expansion. But it is difficult to isolate CD45RA+ Tregs from adult peripheral blood, as the vast majority (>80%) of FOXP3+ T cells are CD45RA− memory cells. Another concern is that contaminating CD4+CD45RA+FOXP3− cells are highly susceptible to conversion to FOXP3+ non-Tregs by TGF-β in the culture medium used for expansion.
Thus, better methods for the isolation and expansion of Tregs that produce greater yields and purity, while maintaining regulatory T cell function, is needed before the treatment of autoimmunity and transplant rejection by adoptive immunotherapy will be viable.